Cdc1p is an endoplasmic reticulum-localized putative lipid phosphatase that affects Golgi inheritance and actin polarization by activating Ca2+ signaling

In the budding yeast Saccharomyces cerevisiae, mutations in the essential gene CDC1 cause defects in Golgi inheritance and actin polarization. However, the biochemical function of Cdc1p is unknown. Previous work showed that cdc1 mutants accumulate intracellular Ca(2+) and display enhanced sensitivity to the extracellular Mn(2+) concentration, suggesting that Cdc1p might regulate divalent cation homeostasis. By contrast, our data indicate that Cdc1p is a Mn(2+)-dependent protein that can affect Ca(2+) levels. We identified a cdc1 allele that activates Ca(2+) signaling but does not show enhanced sensitivity to the Mn(2+) concentration. Furthermore, our studies show that Cdc1p is an endoplasmic reticulum-localized transmembrane protein with a putative phosphoesterase domain facing the lumen. cdc1 mutant cells accumulate an unidentified phospholipid, suggesting that Cdc1p may be a lipid phosphatase. Previous work showed that deletion of the plasma membrane Ca(2+) channel Cch1p partially suppressed the cdc1 growth phenotype, and we find that deletion of Cch1p also suppresses the Golgi inheritance and actin polarization phenotypes. The combined data fit a model in which the cdc1 mutant phenotypes result from accumulation of a phosphorylated lipid that activates Ca(2+) signaling.     

RNA–protein binding kinetics in an automated microfluidic reactor

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.     

Integrating systemic and molecular levels to infer key drivers sustaining metabolic adaptations

Metabolic adaptations to complex perturbations, like the response to pharmacological treatments in multifactorial diseases such as cancer, can be described through measurements of part of the fluxes and concentrations at the systemic level and individual transporter and enzyme activities at the molecular level. In the framework of Metabolic Control Analysis (MCA), ensembles of linear constraints can be built integrating these measurements at both systemic and molecular levels, which are expressed as relative differences or changes produced in the metabolic adaptation. Here, combining MCA with Linear Programming, an efficient computational strategy is developed to infer additional non-measured changes at the molecular level that are required to satisfy these constraints. An application of this strategy is illustrated by using a set of fluxes, concentrations, and differentially expressed genes that characterize the response to cyclin-dependent kinases 4 and 6 inhibition in colon cancer cells. Decreases and increases in transporter and enzyme individual activities required to reprogram the measured changes in fluxes and concentrations are compared with down-regulated and up-regulated metabolic genes to unveil those that are key molecular drivers of the metabolic response.     

Sec16 influences transitional ER sites by regulating rather than organizing COPII

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.     

Early events of the exogenously provided L-Carnitine in murine macrophages,T- and B-lymphocytes: modulation of prostaglandin E1 and E2 production in response to arachidonic acid

L-carnitine is an essential energy-providing compound to the cell since it transports long chain fatty acids through the mitochondrial membrane and delivers them to the beta-oxidation pathway for catabolism and/or entrance to biosynthetic pathways. Some of the early events taking place in immune cells after L-carnitine inoculation in vitro are defined in this report. Using arachidonic acid as a fatty acid source, we determined the utilization rate of L-carnitine by murine T-, B-lymphocytes and macrophages within two hours of cell culture, its effect on prostaglandin E1 and E2 production and the levels of beta-hydroxy-butyrate. The results show that although all immune cells consume a small portion of L-carnitine, beta-hydroxy-butyrate decreases upon addition of arachidonic acid and/or L-carnitine indicating that active biosynthetic pathways are induced. L-carnitine is shown to increase the arachidonic acid-induced production of prostaglandins E1 and E2 in macrophages, while their secretion from T- and B-lymphocytes is decreased. These findings indicate the L-carnitine may very rapidly alter the activation state of immune cells and lead to the development of various reactions, beneficial or not to the organism.     

Escherichia coli SecA truncated at its termini is functional and dimeric

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.     

Purification of a functional mature region from a SecA-dependent preprotein

Most of the bacterial proteins that are active in extracytoplasmic locations are translocated through the inner membrane by the Sec translocase. Translocase comprises a membrane "pore" and the peripheral ATPase SecA. Where preproteins bind to SecA and how they activate translocation ATPase remains elusive. To address this central question we have purified to homogeneity the mature and preprotein parts of an exported protein (pCH5EE). pCH5EE satisfies a minimal size required for protein translocation and its membrane insertion is SecA-dependent. Purified pCH5EE and CH5EE can form physical complexes with SecA and can functionally suppress the elevated ATPase of a constitutively activated mutant. These properties render pCH5EE and CH5EE unique tools for the biochemical mapping of the preprotein binding site on SecA.     

Differentiation between responses of primary productivity and phosphorus exploitation to species richness

Several studies have shown that ecosystem functioning increases with increasing species richness. Most of these studies examined the effects of species richness on primary productivity. The underlying mechanism that explains this pattern is usually the selection effect. The higher the diversity in plant communities the higher the chance in including a very productive species that dominates the community, or a legume species that brings N into the soil. Less attention has been given so far to the effects of species richness on phosphorus exploitation. The aim of this work was to investigate the effect of species richness on aboveground primary productivity and P accumulation in a plant diversity experiment. For this reason, 14 grassland plant species were grown in containers as monocultures and in mixtures of 2-, 3-, 4-, 8-, 11- and 14-species combinations. Results show that the aboveground phytomass and total P increased with increasing species richness. Complementarity effects, probably through partitioning of resources, were most apparent in the highest levels of species richness, and were observed to be greater for total P in comparison to phytomass. Selection effects generally were greater for phytomass than for total P; they were significantly positive at the 2- to 8-species combinations but close to 0 or negative in the highest levels of species richness. The increases in phytomass and total P at the highest levels of species richness appeared to be caused by the increased performance of intermediate-productive species. Responsible Editor: Tibor Kalapos.     

Functional groups' performances as influenced by nitrogen,phosphorus and nodule inhibition of legumes

Aims Nitrogen (N) and phosphorus (P) constitute essential elements for plant growth and their availability influence species diversity in herbaceous plant communities. Legumes exhibit relatively high abundance in N-limited soils. Moreover, the legumes' N:P ratios are much higher than those of the other plant species grown in the same site, probably because they are able to fix atmospheric N 2. The objective of this study was to determine how the relative proportion in N and P availability and the restriction of legumes to fix atmospheric N 2 affect: (i) the primary productivity of plant species, (ii) species composition and (iii) N and P concentrations of species.Methods In an outdoor experiment, mixtures containing grasses, legumes and non-legume forbs were established in 32 containers under four soil treatments (control, N addition, P addition and disinfected soil), in a completely randomized design with eight replicates. Plant growth was examined when N and P were limited in the control soil:sand mixture, in a pot experiment sown with Plantago lanceolata .Important findings The pot experiment indicated that both N and P were limiting for the growth of P. lanceolata. Soil treatments affected primary productivity and species composition. Legumes had a relatively high abundance in the control and their growth was favoured, especially that of Medicago sativa, by P addition. Grasses' growth was increased by the addition of N. Inhibition of rhizobia resulted in poor growth of legumes and concomitant higher growth of grasses, in comparison to the control. The N:P ratios of non-legume species differed between treatments and were always higher in the legume species, even in the disinfected soil. The latter provides evidence that the high N concentrations found in legumes are a physiological characteristic of this specific group of plants.     

Identification of the preprotein binding domain of SecA

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.